Recent research has suggested that molecules highly related to erythrocyte spectrin may be involved in connecting actin filaments to the plasma membrane in a variety of cell types. The goal of the proposed research is to analyze the structure of these putative connecting molecules and elucidate their role in membrane-microfilament interactions. Our previous work suggested that a number of distinct spectrin-like molecules existed. The proposed research will address the question of how many different spectrin -related molecules there may be. Similarities and differences between different members of this class of molecules will be analyzed using biochemical, immunological and electron microscopic tools. Chemical "domains" of these proteins will be generated by limited tryptic digestion, and the tryptic fragments analyzed by two dimensional peptide mapping as well as by reactivity with monoclonal and conventional antibodies. Rotary metal shadowing electron microcopy of antibodies bound to spectrin-like proteins will allow functional domains to be "mapped" on the double stranded, elongated structure. The tissue, cellular and subcellular distribution of individual members of this class of proteins will be investigated by immunofluorescence microscopy and immunoblotting techniques. The manner in which spectrin-like proteins are attached to the plasma membrane will be studied, in addition to possible regulation of their structure or function by calcium, calmodulin or phosphorylation. Alterations in the amount, distribution or function of these proteins when cells are malignantly transformed will be explored as well. This research should yield important insights into membrane-microfilament connections, an area thus far ill-defined in terms of molecules and specific interactions. Since interaction of the plasma membrane with microfilaments is thought to be crucial to many important cellular functions such as cell shape and deformability, cell movement, adhesion and cytokinesis and intracellular mobility, this research should help us understand how these functions are mediated in normal and malignant cells.